Psoralen and psoralen derivatives are polycyclic planar molecules which posses an affinity for nucleic acids, both deoxyribo- and ribo-nucleic acids. See Song, P-S and Tapley, K. J., Photochem Photobiol 29, 1177 (1979) and Scott, B. R., et al., Mutation Research 39, 29 (1976). The structure of psoralen is ##STR1## Specific psoralen derivatives include, among others, 8-methoxypsoralen, 4,5',8-trimethylpsoralen (trioxsalen), 4'-methoxymethyl-4,5',8-trimethylpsoralen, 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) and 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT). As used hereinafter, psoralen or psoralens will be used to refer to both psoralen and psoralen derivatives unless indicated otherwise.
Psoralen is added covalently to a pyrimidine base by a photochemical reaction when a psoralen-nucleic acid complex is irradiated by long wave length ultraviolet light (LWUV). Cole, R. S., Biochem, Biophys Acta 217, 30 (1970) and Musajo, L., et al., Photochem Photobiol 6, 711 (1967). LWUV is ultraviolet light generally having a wavelength of 320-380 nm. Hanson, C. V., et al., J. Gen. Virol. 40, 345 (1978), Hearst, J. E. and Thiry, L., Nucleic Acids Research 4, 1339 (1977) and Musago, L., et al., Experientia 21, 22 (1965) have shown that DNA viruses and RNA viruses are inactivated, i.e. they have lost their ability to infect, by treating the viruses with psoralens and irradiation with LWUV. It has been found that AMT and HMT are the most effective. Hanson et al., also set forth at pages 346-7 numerous advantages of psoralens which include their ability to readily pass through cellular and nuclear membranes and their non-reactivity in the absence of irradiation with LWUV. At page 345, Hanson et al., have further suggested that they would not expect treatment of viruses with psoralens and irradiation with LWUV to cause modification of surface antigens. Thus, they expect the viruses to maintain their antigenicity.
Although psoralens have been utilized for treating viruses, the effects of psoralens on virus-infected cells has not been studied. It was not known whether psoralens would inactivate virus-infected cells so that they would lose their infectivity. It was also not known how psoralen treatment would affect the antigenicity of the virus-infected cells. If psoralen treatment would inactivate virus-infected cells but would not affect their antigenicity, these cells could then be useful for in vitro assays of cellular immunity.
In the past, in vitro assays of virus-specific cellular immunity generally involved the exposure of viable effector lymphocytes to viral antigens, followed by the measurement of some presumably relevant cellular response, such as proliferation, lymphokine release, or cytotoxicity. See Shillitoe, E. J. and Rapp, R., Springer Sem. Immunopathol 2, 237 (1979) and Sissons, J. G. P. and Oldstone, M. B. A., J. Infect. Dis. 142, 114 (1980). Because virus infection of the effector lymphocytes may alter these measured responses, the virus antigens employed should be noninfectious. Plaeger-Marshall, S. and Smith J. W., J. Inf. Dis. 138, 506 (1978); Rosenberg, G. L., et al., Proc. Nat. Acad Sci USA 69, 756 (1972); and, Russell, A. S., Am. J. Clin. Path. 60, 826 (1973). Moreover, since in vivo cell-mediated immunity to many viruses, especially to members of the Herpes virus group, may involve the interaction of effector lymphocytes with virus-infected cells rather than with cell-free virus or soluble antigens (Notkins, A. L., J.Cell Immunol., 11, 478 (1974)), it may be important to use antigen-bearing virus-infected cells rather than cell-free virus or soluble virus antigens in in vitro assays of cellular immunity. Thus, inactivated, virus-infected cells having antigenicity but no infectivity would be useful in these circumstances. The present invention describes the inactivation of virus-infected cells by treatment of these cells with psoralens and irradiation with LWUV. These inactivated, virus-infected cells were found to be antigenic and not infective. These cells are useful for in vitro assays of cellular immunity and as target antigens in serologic assays. Diagnostic kits containing the inactivated cells are prepared for such uses.
Inactivation of target cells with psoralen and irradiation with LWUV have the following advantages: it eliminates the infectivity of the cells in a much shorter time, requires low amounts of radiant energy, permits the recovery of essentially all of the cells initially present and preserves native antigenicity by all criteria examined.